CN109825444A - A kind of bacterial strain and its brewing method improving red rice yellow wine food safety - Google Patents

Abstract

The invention belongs to food brewing technical fields, and in particular to a kind of bacterial strain and its brewing method for improving red rice yellow wine food safety.That the present invention provides a kind of citrinin synthesis capabilities is low, produces high bacterial strain --- the purple Monascus CGMCC No.16790 of amylase activity, liquid melody is prepared using the bacterial strain pure-blood ferment, the brewing of red rice yellow wine is carried out in conjunction with the dry saccharomyces cerevisiae of activity, considerably reduce other unknown microorganisms in brewing process, the food safety of brewing red rice yellow wine is improved, obtained red rice yellow wine color is orange red, full of nutrition, excellent flavor.

Description

A kind of bacterial strain and its brewing method improving red rice yellow wine food safety

Technical field

The invention belongs to food brewing technical fields, and in particular to it is a kind of improve red rice yellow wine food safety bacterial strain and its Brewing method.

Background technique

Yellow rice wine be the big brewed wine of Chinese history brewed wine most remote and the world three (yellow rice wine, grape wine and beer) it One.Brewing yellow rice wine is the fermentation process of an exploitation, and the microorganism that brewing process participates in is from distiller's yeast, raw material, round And air etc., there are absidia corymbifera, aspergillus fumigatus, the more born of the same parents bacterium of sugar, bacillus, staphylococcus, thermophilic actinomycete and enterobacterias Deng (Lu Jian, Cao Yu, Fang Hua, Li Wangjun, Xie Guangfa, Zou Huijun, the Primary Study of fungi in Hu Zhiming's shao-hsing rice wine wheat koji Food and biotechnology journal, 2008,2(2): 78-83；Liu Yunya, Mao Jian, Meng Xiangyong, try to gain the stalks of rice, wheat, etc., and Ji Zhongwei, Feng Dongyang consolidate Bacterial quorum sensing analysis Chinese food journal in red shao-hsing rice wine wheat koji and fermentation process, 2017,17(1): 201-207) Microbe species are various and changeable, and while completing the fermentation of yellow rice wine product, there is also apparent food safety risks.

Red rice yellow wine is the characteristic yellow rice wine that Zhejiang and Fujian some areas use red yeast rice brewing, is rich in Lip river in red yeast rice and cuts down him The active material beneficial to human body such as spit of fland, γ-aminobutyric acid, the product using red yeast rice preparation have good health-care efficacy.Specially Sharp CN 104059831 discloses a kind of fermented type red rice yellow wine and its production method rich in Lovastatin；Patent CN 102885303 disclose a kind of high-content r- aminobutyric acid highland barley monascus and preparation method thereof.But common Monascus is being grown Citrinin can be generated in metabolic process, animal experiments show that citrinin has significant Toxicity of Kidney, Jiangxi honor etc. is each from China perhaps Ground has collected 36 red yeast rice samples, has detected citrinin therein, it is found that (Jiangxi is flourish perhaps, and Lu Chen is solemn containing citrinin in 35 samples Dawn is green, Chen Jiliang, Chen Yun, Wu Yanping, Gu Yumei, and Wu Miaoye part monascus strain produces the Wuxi the research light industry of citrinin College journal, 2000,19(1): 58-61).Therefore, breeding does not produce citrinin but the high red yeast rice bacteria strain of amylase activity, system Standby purebred liquid melody makes red rice yellow wine, can effectively improve the food safety of red rice yellow wine.

Summary of the invention

The purpose of the invention is to overcome shortcoming and defect of the existing technology, and provide a kind of raising red rice yellow wine The bacterial strain and its brewing method of food safety.

The technical solution used in the present invention is as follows: a kind of bacterial strain improving red rice yellow wine food safety, deposit number Are as follows: CGMCC No. 16790, purple Monascus Monascus purpureus.

Above-mentioned strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms on November 26th, 2018 Center (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode: 100101.The strain name of the bacterial strain is: WZ-37；Classification naming is purple Monascus (Monascus purpureus)；Preservation Number are as follows: CGMCC No. 16790.

A kind of red yeast rice improving red rice yellow wine food safety, is prepared by following procedure: by above-mentioned raising red rice yellow wine The strain inoculated of food safety shaking table culture 72 hours into seed culture fluid, revolving speed 180rpm 30 DEG C of temperature, will be cultured Seed liquor in fermentation culture, is placed in shaking table culture, revolving speed 180rpm, temperature by the inoculum concentration transferred species of 10% percent by volume 30 DEG C, culture put bottle after 96 hours, obtained the liquid red yeast rice rich in amylase.

The red yeast rice being often used on existing market is included there are many strain, complicated, various and uncontrollable, therefore the present invention mentions A kind of red yeast rice is supplied, wherein containing only a kind of bacterial strain, it is higher which produces amylase activity, it is sufficient to meet required for yellow rice wine brewage Condition, and citrinin synthesis capability is low, during manufacturing red yeast rice and brewing yellow rice wine, will not synthesize citrinin, institute substantially Simultaneously no citrinin is detected by the method that existing citrinin detects in the yellow rice wine of brewing.

A kind of brewing method of red rice yellow wine, comprising the following steps:

(1) preparation of purebred liquid red yeast rice: by the strain inoculated of above-mentioned raising red rice yellow wine food safety to seed culture fluid Middle shaking table culture 72 hours, revolving speed 180rpm, 30 DEG C of temperature, cultured seed liquor is pressed to the inoculum concentration of 10% percent by volume Transferred species is placed in shaking table culture in fermentation culture, and revolving speed 180rpm, 30 DEG C of temperature, culture put bottle after 96 hours, was rich in The liquid red yeast rice of amylase；

(2) by rice in steep, cook, cool down, in the ratio of 3:1-1:3 rice is added in sterilization container and rich in amylase Liquid red yeast rice adds the 0.05% dry saccharomyces cerevisiae of activity, and gauze sealing, 28 DEG C ferment 10-20 days, through filtering, sterilizing, old It makes, obtains red rice yellow wine.

The fermentation culture is tapioca starch, coarse rice powder, polished rice powder, glutinous rice flour or the wheat flour of 8-15%.

Beneficial effects of the present invention are as follows: that the present invention provides a kind of citrinin synthesis capabilities is low, it is high to produce amylase activity Bacterial strain --- purple Monascus CGMCC No.16790 prepares liquid melody using the bacterial strain pure-blood ferment, make in conjunction with activity is dry Brewer yeast carries out the brewing of red rice yellow wine, considerably reduces other unknown microorganisms in brewing process, improves brewing red yeast rice The food safety of yellow rice wine, obtained red rice yellow wine color is orange red, full of nutrition, excellent flavor.

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention, for those of ordinary skill in the art, without any creative labor, according to These attached drawings obtain other attached drawings and still fall within scope of the invention.

Fig. 1 is using MEGA7.0 software, and ortho position connection method shows bacterial strain CGMCC No. 16790 and GeneBank data Related kind of 18S rDNA sequential system develops tree in library.

Specific embodiment

To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing Step ground detailed description.

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.

Bacterium culture medium used in following embodiment is as follows:

Slant medium (g/L): potato 200(liquor), glucose 20, agar 20, pH nature；

Seed culture medium (g/L): yellow bean sprout 200(liquor), glucose 20, pH nature；

Fermentation medium (g/L): rice meal 150, pH are natural.

The acquisition of 1 strain of embodiment

(1), starting strain

Purple Monascus (Monascus purpureus) is separated from the brewing yellow rice wine red yeast rice that Wenzhou Area is collected and is obtained.

(2), the incubation of strain

Starting strain Monascus is inoculated in slant medium, 30 DEG C of culture monascuses use sterile saline to spore maturation Lower spore is washed, is transferred in the triangular flask with bead, is vibrated, is sufficiently broken up spore, prepare spore suspension.

(3), ultraviolet mutagenesis

Spore suspension is dispensed into sterile petri dish, by the ultraviolet lamp that spore suspension is placed in wavelength 254nm, power is 40W Lower irradiation carries out mutagenic treatment.Gradient dilution after processing, and it is coated on slant medium, it is cultivated 3 days in 30 DEG C.

(4), NTG mutagenic treatment

The NTG of 2mg/mL is added in spore suspension, in oscillation treatment 20-60 minutes on 30 DEG C of shaking tables, sampling gradient dilution was applied Cloth plate separates inclined-plane, cultivates 3 days in 30 DEG C.

(5), shaking flask is screened

The single colonie handled through breeding is inoculated in slant medium, 30 DEG C are cultivated 3 days, be inoculated in seed culture medium and 30 DEG C of fermentation medium cultures, analyze carbohydrase enzyme activity and citrinin content.Take glucoamylase enzyme vigor high and citrinin content The bacterial strain of low (or being free of citrinin) after plate separates and after fermented and cultured verifying, submits preservation, and microorganism fungus kind protects Hiding number is CGMCC No.16790, and strain improvement pedigree is shown in Fig. 1.

(6), saccharifying enzymic activity measures

8000 r/min of fermentation liquid is centrifuged 10 min, supernatant is taken to measure carbohydrase using 3,5- dinitrosalicylic Acid Colorimetry Vigor.Saccharifying enzymic activity definition: under conditions of 40 DEG C, 5.6 pH, 1 h hydrolyzes soluble starch and generates 11 mL enzyme solution Mg glucose is 1 enzyme activity unit (U/mL).

(7), the measurement of citrinin content

8000 r/min of fermentation liquid is centrifuged 10 min, takes supernatant with 0.45 μm of membrane filtration.It is measured using HPLC, liquid Phase chromatographic condition: C18 chromatographic column (250 mm × 4.6 mm, 5 μm)；The excitation wavelength of fluorescence detector is λ ex=331 nm, Emission wavelength lambda em=500 nm；Mobile phase is V(acetonitrile): V(water)=35: 65, phosphorus acid for adjusting pH 2.5；28 DEG C of column temperature；Stream 1.2 mL/min of speed；20 μ L(specific method reference of sample volume: Yue Jianming, Yang Qiang, Xiao Xiao wait ammonium salt to close monascus parpureus Went At metabolism monascorubin and citrinin influence Food Science, 2016,37(5): 102-107).

The morphological feature of 2 purple Monascus CGMCC No. 16790 of embodiment

In PDA culture medium culture 5-10 days, morphological feature was that mycelium initial stage is colourless, faded to red, and become culture medium Aubergine.The a large amount of branches of mycelium generate single or bunchiness conidium, spore on the top of branch containing the purplish red coloured particles of orange Son is spherical in shape or oval, and (6.5 μm -10.5 μm) × (7 μm ~ 9 μm), cleistothecium is orange red, subsphaeroidal, contains most ascus, Ascus includes 8 spores, and 25 μm -75 μm of diameter, ascospore is oval or subsphaeroidal, smooth, transparent, colourless or Qi Hongse, (5.5 μm -6 μm) × (3.5 μm -5 μm) illustrate that the bacterial strain is purple Monascus Monascus purpureus.

3 purple Monascus CGMCC No. of embodiment, 16790 strain 18SrDNA identification

The genomic DNA of purple Monascus CGMCC No. 16790 is extracted, 18S rDNA analysis is carried out.

Design primer, forward primer: 5 '-GCATCACA- of 5 '-GTAGTCATATGCTTGTCTC-3 ' and reverse primer GACCTGTTATTGCCTC-3'；PCR amplification is carried out, PCR reaction system composition is as follows:

PCR condition is as shown in the table:

18S rDNA in DNA sequencing, with NCBI Genbank database is carried out to PCR product and carries out sequence alignment, as a result See Fig. 1.It can be seen that the 18SrDNA phase of purple Monascus CGMCC No. 16790 bacterial strain and Monascus purpureus Together, purple Monascus can be accredited as.

The fermented and cultured of 4 shaking flask of embodiment

Purple Monascus CGMCC No. 16790 is inoculated in slant medium, 30 DEG C are cultivated 3 days.Inclined-plane thalline is inoculated with In the 250ml triangle shake bottle of the seed culture fluid containing 100ml, it is placed in shaking table culture 72 hours, revolving speed 180rpm, 30 DEG C of temperature. Cultured seed liquor is set in the 500ml triangle shake bottle of the fermentation culture containing 150ml by 10% (v/v) inoculum concentration transferred species In shaking table culture, revolving speed 180rpm, 30 DEG C of temperature.Bottle is put in culture after 96 hours, detection carbohydrase enzyme activity is 116.7U/ml, tangerine Mycin is not detected.

The brewing of 5 yellow rice wine of embodiment

1, the preparation of purebred liquid red yeast rice: purple Monascus CGMCC No. 16790 is activated 3 days on PDA slant medium, Then it accesses in seed culture medium shaking table culture 72 hours, revolving speed 180rpm, 30 DEG C of temperature.Cultured seed liquor is pressed 10% (v/v) inoculum concentration transferred species shaking table culture in fermentation culture, revolving speed 180rpm, cultivate 96 hours, obtain richness by 30 DEG C of temperature The liquid red yeast rice of amylase-containing；

2, the brewing of yellow rice wine: by rice in steep, cooking, cool down, in sterilized triangular flask in the ratio of 3:1 be added rice and Liquid red yeast rice rich in amylase adds the 0.05% dry saccharomyces cerevisiae of activity, and gauze sealing, 28 DEG C ferment 20 days, pass through Filter, sterilizing, ageing, obtain sapid red rice yellow wine, citrinin is not detected.

The brewing of 6 yellow rice wine of embodiment

1, the preparation of purebred liquid red yeast rice, with embodiment 5；

2, the brewing of yellow rice wine: by rice in steep, cooking, cool down, in sterilized triangular flask in the ratio of 2:1 be added rice and Liquid red yeast rice rich in amylase adds the 0.05% dry saccharomyces cerevisiae of activity, and gauze sealing, 28 DEG C ferment 15 days, pass through Filter, sterilizing, ageing, obtain sapid red rice yellow wine, citrinin is not detected.

The brewing of 7 yellow rice wine of embodiment

1, the preparation of purebred liquid red yeast rice, with embodiment 5；

2, the brewing of yellow rice wine: by rice in steep, cooking, cool down, in sterilized triangular flask in the ratio of 1:3 be added rice and Liquid red yeast rice rich in amylase adds the 0.05% dry saccharomyces cerevisiae of activity, and gauze sealing, 28 DEG C ferment 10 days, pass through Filter, sterilizing, ageing, obtain sapid red rice yellow wine, citrinin is not detected.

The above disclosure is only the preferred embodiments of the present invention, cannot limit the right model of the present invention with this certainly It encloses, therefore equivalent changes made in accordance with the claims of the present invention, is still within the scope of the present invention.

Claims (4)

1. a kind of bacterial strain for improving red rice yellow wine food safety, it is characterised in that: its deposit number are as follows: CGMCC No. 16790, Purple Monascus Monascus purpureus.

2. a kind of red yeast rice for improving red rice yellow wine food safety, which is characterized in that prepared by following procedure: by claim 1 The strain inoculated of the raising red rice yellow wine safety shaking table culture 72 hours, revolving speed 180rpm, temperature into seed culture fluid 30 DEG C, cultured seed liquor is placed in shaking table culture in fermentation culture by the inoculum concentration transferred species of 10% percent by volume, Revolving speed 180rpm, 30 DEG C of temperature, culture put bottle after 96 hours, obtained the liquid red yeast rice rich in amylase.

3. a kind of brewing method of red rice yellow wine, it is characterised in that the following steps are included:

(1) preparation of purebred liquid red yeast rice: the strain inoculated described in claim 1 for improving red rice yellow wine safety is trained to seed Shaking table culture 72 hours in nutrient solution, revolving speed 180rpm, 30 DEG C of temperature, by cultured seed liquor connecing by 10% percent by volume Kind amount transferred species is placed in shaking table culture in fermentation culture, and revolving speed 180rpm, 30 DEG C of temperature, culture put bottle after 96 hours, obtained Liquid red yeast rice rich in amylase；

(2) by rice in steep, cook, cool down, in the ratio of 3:1-1:3 rice is added in sterilization container and rich in amylase Liquid red yeast rice adds the 0.05% dry saccharomyces cerevisiae of activity, and gauze sealing, 28 DEG C ferment 10-20 days, through filtering, sterilizing, old It makes, obtains red rice yellow wine.

4. the brewing method of red rice yellow wine according to claim 3, it is characterised in that: the fermentation culture is 8- 15% tapioca starch, coarse rice powder, polished rice powder, glutinous rice flour or wheat flour.